Principal Investigator/Program Director: Schmidt, Holger Project Summary Spurred by the explosive growth in personalized medicine which requires highly sensitive genomic and proteomic analysis, single molecule detection is rapidly developing from a fundamental research technique into the foundation for advanced molecular diagnostic techniques. Whereas optical fluorescence detection used to be the approach of choice, electrical detection of single molecules as they are drawn through a nanopore has recently gained great attention, particularly in the context of next generation sequencing. The direct, label-free detection of a wide range of molecules using nanopores has much broader potential. However, a major obstacle for fully exploiting the exquisite sensitivity of nanopore detection is the mismatch between the minute electrostatic capture volume of a nanopore and the low concentrations encountered in real-world applications, e.g. in molecular diagnostics. The goal of this project is to overcome this limitation with a novel, innovative approach to high- throughput molecular diagnostics using single molecule nanopore analysis. The specific objectives of this application are to increase the capture rate (throughput) of a nanopore by orders of magnitude and to validate this approach with clinical samples for Zika virus (ZIKV) infection. Our central hypothesis is that this can be accomplished by delivering target molecules isolated on microbeads within the capture radius of the pore using a chip-based optical trap on a waveguide-based optofluidic chip. The objectives of this application will be accomplished by the following Specific Aims: (1) Nanopore capture rate enhancement using optical trapping of carrier beads. This Aim will validate the core of our new approach and demonstrate a 50,000x improvement for both nucleic acids and proteins; (2) On-chip integration of microfluidic sample processing with high throughput molecular detection at ultralow (attomolar) concentrations; and (3) Multiplex direct detection of ZIKV infection starting from complex sample matrices (serum, saliva, urine, semen). The innovative contributions of the proposed work are: (i) Optical trapping of carrier particles for molecular target delivery; (ii) Creating an integrated chip-based system for nanopore-based, label-free detection of diverse molecular biomarkers; and (iii) Clinical validation of nanopore- based analysis of multiple analyte types. The proposed work is significant because it will introduce the first integrated system that matches the sensitivity, simplicity, and versatility of single-molecule nanopore detection with the real-world constraints for molecular diagnostics. As such, this approach will be applicable to many molecular targets and applications. 1